Hepatocyte growth factor (HGF) was discovered as a potent growth factor for mature hepatocytes. A gene encoding the HGF has been cloned and its amino acid sequence has also been revealed (Biochem. Biophys. Res. Com., 122:1450 (1984); Proc. Natl. Acad. Sci. USA, 83:6489, (1986); FEBS Lett., 224:311 (1987); Nature, 342:440 (1989); Proc. Natl. Acad. Sci. USA, 87:3200 (1990)). Through diverse studies, it has been revealed that HGF not only functions as a liver regenerating factor in the repair and regeneration of hepatopathy, but it also possesses various pharmacological effects. Therefore, HGF is expected to serve as a therapeutic agent for renal diseases as well as hepatopathy. In fact, intraperitoneal administration of HGF is shown to exhibit a therapeutic effect for renal fibrosis through the suppression of transforming growth factor-β (TGF-β), a potent fibrogenic factor in mice that spontaneously develop nephrosis (Kidney Int., 57:937 (2000)).
Currently, most important issue in kidney transplantation is abolition of renal function (renal death) due to chronic rejection. It has been demonstrated that such chronic rejection is induced by damage such as ischemia, caused at the time of transplantation (Transplantation, 64:190 (1997)). In Japan, for cadaver-donor kidney transplantation, kidneys are removed after cardiac arrest. This results in prolonged ischemia, leading to chronic rejection and poor long-term prognosis of renal functions. On the other hand, in the case of living donor kidney transplantation, since more than half of all donors are 60 years or older, these kidneys have less reserve force which leads to chronic rejection.
HGF is expected to serve as a novel therapeutic agent aiming to arrest abolition of renal function due to chronic rejection and improving the survival rate of transplanted kidney. About half of all transplanted kidneys are abolished after 32 weeks in Lewis rats transplanted with kidneys from Fisher rats, a rat model for chronic rejection of transplanted kidney. However, when HGF was intravenously administered daily for 4 weeks after transplantation to the model rats, all of the transplanted kidneys survived up to 32 weeks without additional HGF administration. Namely, HGF is known to have the action of protecting transplanted kidneys (Transplant Proc., 31:854 (1999)). This report is significant in showing that suppression of initial damages after transplantation by HGF improves the survival rate of transplanted kidneys without further treatment.
In general, proteins are administered by systemic routes such as intravenous injection. However, HGF protein has a short half-life in blood. Moreover, when administered systemically, the diverse pharmacological actions of HGF are suspected to cause not a little influence on other organs. Therefore, the most effective strategy for reacting HGF on kidney may be methods wherein HGF can be locally and at the same time continuously reacted on the kidney. One way that could be considered to solve this problem would be to introduce an HGF gene only into the kidney to continuously and locally react HGF on the kidney. Since HGF is quickly metabolized in the liver as compared to other methods wherein the HGF gene is introduced into other tissues (such as muscle), and reacting HGF protein produced therein on the kidney, this method is considered advantageous in that: (1) only a little amount of the HGF gene is required for introduction; and (2) the influence on other organs can be reduced. In addition, gene transfer has merit in that it does not require multiple steps for purification, generally as needed for protein drugs. Although continuous injection of protein drugs has merit in that a constant concentration can be maintained in blood, it requires the attachment of a catheter for a long term, which involves results in complex manipulation and risk of infection as well as restraint on patient's movement.
Several methods have been reported for gene transfer targeting kidney, including the method that utilizes the HVJ-liposome method for the injection of a gene through the renal artery as reported by the present inventors (J. Clin. Invest., 92:2597 (1993); Kidney Int., 50:148 (1996)) and the kidney perfusion method utilizing adenovirus vectors (Gene Ther., 3:21 (1996)). However, the safety issue remains to be solved for these methods due to their use of virus vectors for gene transfer. On the other hand, while electroporation has been conventionally used as an in vitro gene transfer method, recently, its use as an effective method for in vivo gene transfer has been reported (Nat. Biotechnol., 16:867 (1998)). However, according to this previously reported method, genes are locally injected into a muscle or a tumor and electric stimuli is applied near the injected site with needle-type electrodes. However, this method has certain disadvantages, such as tissue damage. In addition, gene expression is limited to the injected site and thus it is impossible to express the desired gene in the whole of the transplanted kidney. On the other hand, a gene therapy method utilizing HGF wherein the HGF gene is introduced into a muscle of a rat liver cirrhosis model using HVJ liposome has been reported to exert a therapeutic effect on liver cirrhosis (Nat. Med., 5:226 (1999)). This method was also reported to show a therapeutic effect on a balloon injury model (Gene Ther., 7:1664 (2000)). However, the introduction of an HGF gene into a transplanted kidney to examine the survival rate has not been reported nor is there any report of the effectiveness of ex vivo gene transfer via electroporation.